Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The overall goal of this experiment is to perform multicolor immunohistochemical visualization and enumeration of various T lymphocytes subpopulations in archival formalin-fixed paraffin-embedded tissue sections from oropharyngeal squamous cell carcinoma samples. This method can help answer key questions in immuno-oncology field such as, is there a link between tumor tissue infiltrating immune cells subpopulations and patient prognosis. With this technique, you can visualize several biomarkers in one tissue section.

The implications of this technique extend towards cancer therapy, as the simultaneous evaluation of multiple immune cells or immune related factors might help in patient stratification for specific immune therapies. Begin by using a micro tongue to obtain four micrometer thick formalin-fixed paraffin-embedded tissue sections, laying the tissue ribbons on the surface of a 45 degree celsius de-ionized water bath as they are acquired. To capture the sections, place a glass slide in the water, directly underneath the tissue sample and carefully move the slide up at an angle.

This section will stick to the glass surface as the slide is lifted out of the water. After drying the samples overnight at 37 degrees celsius, de-paraffinize the tissues with three five minute total immersions in 100%xylol. When the sections have been completed de-paraffinized, rehydrate the tissue samples with three descending ethanol immersions.

Followed by a five minute wash in de-ionized water. Then, submerge the samples in boiling EDTA buffer for 10 minutes in the microwave and let the slides cool in the buffer for two hours. For labeling of the antigens of interest, rinse the samples with two five minute PBS washes.

After the second wash, incubate the tissues with the primary antibodies and interest diluted in 1%bovine serum albumin in PBS at room temperature, overnight. The next morning, rinse the sections with three five minute PBS washes and incubate the samples with the appropriate secondary antibodies diluted in 1%serum in PBS at room temperature for one hour. At the end of the incubation, wash the slides three times in PBS as just demonstrated and mount the slides using an aqueous antifade mounting medium containing a nuclear counter stain.

To analyze the samples, first select the 20 to 25 times objective on a fluorescence microscope and capture four images per slide using the appropriate filters for the secondary antibodies and counter stain. Save the images as they are obtained. Next, in the appropriate image analysis software, open the first image of interest and click on the overlay tab.

Select the closed poly line shape to demarquee the boundary of the region of interest. When the shape is closed, the surface area of measurement will appear next to the region of interest. Save all the surface area data in the spreadsheet file to calculate the average surface areas and sound numbers per slide.

Export the image as a TIFF or JPG file to be opened in ImageJ. Then, in ImageJ, open the first image of interest and select the cell counter under the plugins menu. Then, click initialize, click on type one and click on every cell designated as type one.

Select a different type for each cell type to count the number of different types of cells in the image according to the fluorochrome expression. Finally, record the number of cells analyzed in each counter bin for each image in the spreadsheet file. In this representative experiment, on of four random images were analyzed from a series of formalin-fixed paraffin-embedded pretreatment tumor samples obtained from primary oropharyngeal tumors, diagnosed in the Leiden University Medical Center between 1970 to 2011, that were prepared as just demonstrated.

In this experimental sample, some autofluorescent cells were observed, as distinguished by their complete yellow appearance, while negative control slides did not demonstrate any specific staining above background tissue autofluorescence. All of the tumor samples were determined to be infiltrated by varying numbers of fox p3 positive, CD3 positive T cells with only a small T cell subset expressing IL-17. Samples from patients with a high number of total infiltrating CD3 positive T cells demonstrate a trend toward a correlation with an improved disease-free survival compared to patient samples with low T cell members.

Further, in samples with a low number of IL-17 positive cells, a high number of total infiltrating T cells is also correlated with an improved disease-free survival, suggesting that the effective tumor infiltrating T cells in oropharyngeal squamous cell carcinoma maybe related to the low number of IL-17 positive cells present. Once mastered, these sample preparation steps can be completed in one and a half days for a maximum of 25 slides per staining session. It's very important to de-paraffanize the sample, otherwise you'll get a lot of background.

After its development, this technique paved the way for researchers in the fields of pathology and immunology. For exploring the microenvironment for the localisation of various immune cell subpopulations. After watching this video, you should have a good understanding of how to visualize various proteins and cell types in a single archival tissue section.

Don't forget that working with silo is very dangerous, you should always work in a flow cabinet.